DNA enzyme to inhibit plasminogen activator inhibitor-1

ABSTRACT

The present invention provides DNAzymes and ribozymes that specifically cleave PAI-1-encoding mRNA. The present invention also provides antisense oligonucleotides that specifically inhibit translation of PAI-1-encoding mRNA. The invention also provides various methods of inhibiting the expression of PAI-1. Finally the invention provides pharmaceutical compositions containing the instant DNAzymes, ribozymes and antisense oligonucleotides as active ingredients.

[0001] Throughout this application, various publications are referencedin parentheses by arabic numbers. Full citations for these referencesmay be found at the end of the specification immediately preceding theclaims. The disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains.

BACKGROUND

[0002] After a myocardial infarction, the subsequent cardiomyocytenecrosis stimulates a wound healing response characterized by migrationof inflammatory cells into the affected myocardium, extracellular matrixdegradation, and neovascularization. Each of these components appears torequire activation of latent metalloproteinases by plasmin, which isderived from plasminogen through activation by urokinase-typeplasminogen activator (u-PA) expressed on the surface of theinfiltrating bone-marrow derived cells (1-3). More recently, it has beensuggested that the role of cell surface u-PA may be to facilitateexposure of cryptic cell attachment sites necessary for cell migration(4). This appears to involve direct interaction between u-PA andplasminogen activator inhibitor-1 (PAI-1), which, in its native state,is complexed to vitronectin (4,5). Reaction of PAI-1 with a proteinase,such as u-PA, results in a rapid conformational change that causes it todissociate from vitronectin and increase its affinity for the lowdensity lipoprotein receptor (6), leading to its clearance anddegradation. Removal of PAI-1 from vitronectin exposes the epitope onvitronectin necessary for binding to another of its ligands, theintegrin alpha v beta 3 (4,7). Since interactions between the cellsurface integrin alpha v beta 3 and tissue vitronectin have been shownto be important in the development of angiogenesis (4,8,9), wehypothesized that excessive PAI-1 protein expression after myocardialinfarction might prevent optimal neovascularization by bonemarrow-derived angioblasts and that inhibition of PAI-1 expression wouldpromote neovascularization.

[0003] In order to develop an approach to inhibit PAI-1 expression whichwould have clinical applicability, we examined various potentialstrategies for inhibiting specific mRNA activity.

[0004] Antisense oligonucleotides hybridize with their complementarytarget site in mRNA, blocking translation to protein by stericallyinhibiting ribosome movement or by triggering cleavage by endogenousRNAse H (10). Although current constructs are made more resistant todegradation by serum through phosphorothioate linkages, non-specificbiological effects due to “irrelevant cleavage” of non-targeted mRNAremains a major concern (11).

[0005] Ribozymes are naturally-occurring RNA molecules that containcatalytic sites, making them more potent agents than antisenseoligonucleotides. However, wider use of ribozymes has been hampered bytheir susceptibility to chemical and enzymatic degradation andrestricted target site specificity (12).

[0006] A new generation of catalytic nucleic acids has been describedcontaining DNA molecules with catalytic activity for specific RNAsequences (13-16). These DNA enzymes exhibit greater catalyticefficiency than hammerhead ribozymes, producing a rate enhancement ofapproximately 10 million-fold over the spontaneous rate of RNA cleavage,offer greater substrate specificity, are more resistant to chemical andenzymatic degradation, and are far cheaper to synthesize.

SUMMARY

[0007] This invention provides a catalytic nucleic acid thatspecifically cleaves an mRNA encoding a Plasminogen ActivatorInhibitor-1 (PAI-1) comprising, in 5′ to 3′ order:

[0008] (a) consecutive nucleotides defining a first binding domain of atleast 4 nucleotides;

[0009] (b) consecutive nucleotides defining a catalytic domain locatedcontiguous with the 3′ end of the first binding domain, and capable ofcleaving the PAI-1-encoding mRNA at a predetermined phosphodiester bond;and

[0010] (c) consecutive nucleotides defining a second binding domain ofat least 4 nucleotides located contiguous with the 3′ end of thecatalytic domain,

[0011] wherein the sequence of the nucleotides in each binding domain iscomplementary to a sequence of ribonucleotides in the PAI-1-encodingmRNA and wherein the catalytic nucleic acid hybridizes to andspecifically cleaves the PAI-1-encoding mRNA.

[0012] This invention further provides a pharmaceutical compositioncomprising the instant catalytic nucleic acid and a pharmaceuticallyacceptable carrier.

[0013] This invention further provides method of specifically inhibitingthe expression of PAI-1 in a cell that would otherwise express PAI-1,comprising contacting the cell with the instant catalytic nucleic acidso as to specifically inhibit the expression of PAI-1 in the cell.

[0014] This invention further provides a method of specificallyinhibiting the expression of PAI-1 in a subject's cells comprisingadministering to the subject an amount of the instant catalytic nucleicacid effective to specifically inhibit the expression of PAI-1 in thesubject's cells.

[0015] This invention further provides a method of specificallyinhibiting the expression of PAI-1 in a subject's cells comprisingadministering to the subject an amount of the instant pharmaceuticalcomposition effective to specifically inhibit the expression of PAI-1 inthe subject's cells.

[0016] This invention further provides a method of treating acardiovascular disease in a subject involving apoptosis of acardiomyocyte in the subject which comprises administering to thesubject an amount of the instant pharmaceutical composition effective toinhibit apoptosis of the cardiomyocyte in the subject so as to therebytreat the cardiovascular disease.

[0017] This invention further provides a method of treating a fibroticdisease in a subject involving fibrogenesis which comprisesadministering to the subject an amount of the instant pharmaceuticalcomposition effective to inhibit fibrogenesis in the subject so as tothereby treat the fibrotic disease.

[0018] This invention further provides an oligonucleotide comprisingconsecutive nucleotides that hybridizes with a PAI-1-encoding mRNA underconditions of high stringency and is between 8 and 40 nucleotides inlength. This invention further provides the instant oligonucleotide,wherein the human PAI-1-encoding mRNA comprises consecutive nucleotides,the sequence of which is set forth in SEQ ID NO:5.

[0019] This invention further provides a method of treating a subjectwhich comprises administering to the subject an amount of the instantoligonucleotide of effective to inhibit expression of a PAI-1 in thesubject so as to thereby treat the subject.

[0020] This invention further provides a method of treating acardiovascular disease in a subject involving apoptosis of acardiomyocyte in the subject which comprises administering to thesubject an amount of the instant oligonucleotide effective to inhibitapoptosis of the cardiomyocyte in the subject so as to thereby treat thecardiovascular disease.

[0021] This invention further provides a method of treating a fibroticdisease in a subject involving fibrogenesis in the subject whichcomprises administering to the subject an amount of the instantoligonucleotide effective to inhibit fibrogenesis in the subject so asto thereby treat the fibrotic disease.

BRIEF DESCRIPTION OF THE FIGURES

[0022] FIGS. 1(A)-(D): (A) This figure shows three DNA enzymes, termedE1 (SEQ ID NO:2), E2 (SEQ ID NO:4), and E3 (SEQ ID NO:3), containingidentical 15-nucleotide catalytic domains (SEQ ID NO:1) flanked by twoarms of eight nucleotides (E1, E2) or nine nucleotides (E3) withcomplementarity to human (E1, E3) or rat (E2) PAI-1 mRNA. This figurealso shows the control DNA enzyme E0 (SEQ ID NO:7), an oligonucleotideS1 (SEQ ID NO:8), an oligonucleotide transcript (SEQ ID NO:9) andoligonucleotide S2 (SEQ ID NO:10). (B) This figure shows the 21-baseoligonucleotide S1 (SEQ ID NO:8), synthesized from human PAI-1 mRNA andlabeled at the 5′ end with 32P, was cleaved within 2 minutes whencultured with E1 at 10:1 substrate:enzyme excess, with maximal cleavageoccurring by 2 hours. (C) This figure shows E1 also cleaved larger32P-labeled fragments of human PAI-1 mRNA, prepared by in vitrotranscription, in a time- and concentration-dependent manner. (D) Thisfigure shows the sequence-specific nature of the DNA enzymatic cleavage:the control DNA enzyme E0, containing an identical catalytic domain toE1 and E3, but scrambled sequences in the flanking arms, caused nocleavage of human PAI-1 mRNA transcripts.

[0023] FIGS. 2(A)-(D): (A) This figure shows that the DNA enzyme E2cleaved the 23-base oligonucleotide S2 (SEQ ID NO:10), synthesized fromthe sequence of rat PAI-1 mRNA, in a dose- and time-dependent manner.(B) This figure shows that E2 also cleaved a rat PAI-1 mRNA transcriptin a dose-dependent manner by 2-4 hours to give the 156 nucleotidecleavage product. (C) This figure shows the rat S2 PAI-1 mRNA transcript(SEQ ID NO:10) differs by only one nucleotide from the human mRNA PAI-1transcript (SEQ ID NO:9) which can be cleaved by E3.

[0024] FIGS. 3(A)-(D): (A) This figure shows Densitometric analysis ofRT-PCR products following reverse transcription of cellular mRNA: E2inhibited TGF-beta inducible steady-state mRNA levels in cultured ratendothelium by 52% relative to the E0 scrambled DNA.

[0025] (B) This figure shows the effect of endothelial cell transfectionwith E2 DNA enzyme on TGF-beta mediated induction of PAI-1 protein.Endothelial cells transfected with scrambled DNA enzyme demonstratedapproximately 50% increase in cytoplasmic PAI-1 E0 protein as detectedby Western blot. In contrast, this effect was almost completelyabrogated by transfection with the PAI-1 DNA enzyme E2. (C) This figureshows that whereas the DNA enzyme was almost completely degraded within6 hours of cell culture in medium containing 20% serum, it remainedintact during the entire 24 hours of cell culture with medium containing2% serum. (D) This figure shows that HUVEC transfected with E1 or E3 DNAenzymes and cultured for 12 hours in 2% serum demonstrated 20% and 28%reduction in TGF-beta dependent PAI-1 activity compared withtransfection with the scrambled DNA enzyme E0 (both p<0.01).

[0026] FIGS. 4(A)-(C): (A) This figure shows that at two weeks afterinjection with E2, PAI-1 expression in infarcted rat hearts wasdramatically altered, with 71% reduction in PAI-1 expression bymacrophages at the peri-infarct region (p<0.01). (B) and (C) showinjection of E2 and angioblasts increased the number of CD31 positivecapillaries.

[0027] FIGS. 5A-C: (A) This figure shows inhibition of cardiomyocyteapoptosis in the peri-infarct region by E2. (B) This figure shows thepercentage recovery in left ventricular ejection fraction (LVEF) aftertreatment with E2 and after treatment with E2 and angioblasts.

[0028]FIG. 6: This figure shows the DNA sequence encoding humanPlasminogen Activator Inhibitor-1 protein (SEQ ID NO:5).

[0029]FIG. 7: This figure shows the DNA sequence encoding ratPlasminogen Activator Inhibitor-1 protein (SEQ ID NO:15).

[0030]FIG. 8: This figure shows the amino acid sequence of humanPlasminogen Activator Inhibitor-1 protein (SEQ ID NO:6).

[0031]FIG. 9: This figure shows the amino acid sequence of ratPlasminogen Activator Inhibitor-1 protein (SEQ ID NO:16).

[0032]FIG. 10: This figure shows possible 5′-AT-3′ cleavage sites onhuman PAI-1 mRNA (shown as corresponding DNA, SEQ ID NO:5) for catalyticdeoxyribonucleic acids. Bold indicates the protein coding region,capital letters indicate consensus cleavage sites.

[0033]FIG. 11: This figure shows possible 5′-AC-3′ cleavage sites onhuman PAI-1 mRNA (shown as corresponding DNA, SEQ ID NO:5) for catalyticdeoxyribonucleic acids. Bold indicates the protein coding region,capital letters indicate consensus cleavage sites.

[0034]FIG. 12: This figure shows possible cleavage sites on human PAI-1mRNA coding region (shown as corresponding DNA, SEQ ID NO:17) forcatalytic hammerhead ribonucleic acids. Uppercase “T” representscleavage site.

DETAILED DESCRIPTION

[0035] As used herein, and unless stated otherwise, each of thefollowing terms shall have the definition set forth below.

[0036] “Administering” shall mean any of the various methods anddelivery systems known to those skilled in the art. The administeringcan be performed, for example, via implant, transmucosally,transdermally and subcutaneously, orally, parenterally, topically, bycardiac injection, by inhalation, by catheter e.g. retrograde uretericcatheter, by intra-arterial injection.

[0037] “Catalytic” shall mean the functioning of an agent as a catalyst,i.e. an agent that increases the rate of a chemical reaction withoutitself undergoing a permanent structural change.

[0038] “Consensus sequence” shall mean a nucleotide sequence of at leasttwo residues in length between which catalytic nucleic acid cleavageoccurs. For example, consensus sequences include for catalyticdeoxyribonucleic acids are purine:pyrimidine e.g. “A:U” and “G:U”.

[0039] “Plasminogen Activator Inhibitor-1 Protein” shall mean theprotein encoded by the nucleotide sequence identified as (SEQ ID NO:5)and having the amino acid sequence shown in SEQ ID NO:6, when identifiedas human and the protein encoded by the nucleotide sequence identifiedas SEQ ID NO:15 and having the amino acid sequence shown in SEQ ID NO:16when identified as originating from rat, and any variants of eitherthereof, whether artificial or naturally occurring. Variants include,without limitation, homologues, post-translational modifications,mutants and polymorphisms.

[0040] “Plasminogen Activator Inhibitor-1 mRNA” shall mean a mRNAmolecule comprising a sequence which encodes Plasminogen ActivatorInhibitor-1 Protein. Plasminogen Activator Inhibitor-1 mRNA includes,without limitation, protein-encoding sequences as well as the 5′ and 3′non-protein-encoding sequences.

[0041] “Hybridize” shall mean the annealing of one single-strandednucleic acid molecule to another nucleic acid molecule based on sequencecomplementarity. The propensity for hybridization between nucleic acidsdepends on the temperature and ionic strength of their milieu, thelength of the nucleic acids and the degree of complementarity. Theeffect of these parameters on hybridization is well known in the art(see 38).

[0042] “Inhibit” shall mean to slow, stop or otherwise impede.

[0043] “Stringent conditions” or “Stringency”, shall refer to theconditions for hybridization as defined by the nucleic acid, salt, andtemperature. These conditions are well known in the art and may bealtered. Numerous equivalent conditions comprising either low or highstringency depend on factors such as the length and nature of thesequence (DNA, RNA, base composition), nature of the target (DNA, RNA,base composition), milieu (in solution or immobilized on a solidsubstrate), concentration of salts and other components (e.g.,formamide, dextran sulfate and/or polyethylene glycol), and temperatureof the reactions (within a range from about 5° C. below the meltingtemperature of the probe to about 20° C. to 25° C. below the meltingtemperature). One or more factors be may be varied to generateconditions of either low or high stringency different from, butequivalent to, the above listed conditions. As will be understood bythose of skill in the art, the stringency of hybridization may bealtered in order to identify or detect identical or relatedpolynucleotide sequences.

[0044] “Nucleic acid” shall include any nucleic acid, including, withoutlimitation, DNA, RNA, oligonucleotides, or polynucleotides, and analogsor derivatives thereof. The nucleotides that form the nucleic acid maybe nucleotide analogs or derivatives thereof. The nucleic acid mayincorporate non nucleotides.

[0045] “Nucleotides” shall include without limitation nucleotides andanalogs or derivatives thereof. For example, nucleotides may comprisethe bases A, C, G, T and U, as well as derivatives thereof. Derivativesof these bases are well known in the art, and are exemplified in PCRSystems, Reagents and Consumables (Perkin Elmer Catalogue 1996-1997,Roche Molecular Systems, Inc., Branchburg, N.J., USA).

[0046] “Pharmaceutically acceptable carrier” shall mean any of thevarious carriers known to those skilled in the art.

[0047] “Specifically cleave”, when referring to the action of one of theinstant catalytic deoxyribonucleic acid molecules on a target mRNAmolecule, shall mean to cleave the target mRNA molecule without cleavinganother mRNA molecule lacking a sequence complementary to either of thecatalytic deoxyribonucleic acid molecule's two binding domains.

[0048] “Subject” shall mean any animal, such as a human, a primate, amouse, a rat, a guinea pig or a rabbit.

[0049] The terms “DNA enzyme”, “DNAzyme” and “catalytic deoxyribonucleicacid” are used synonymously.

[0050] “Vector” shall include, without limitation, a nucleic acidmolecule that can be used to stably introduce a specific nucleic acidsequence into the genome of an organism.

[0051] This invention provides a catalytic nucleic acid thatspecifically cleaves an mRNA encoding a Plasminogen ActivatorInhibitor-1 (PAI-1) comprising, in 5′ to 3′ order:

[0052] (a) consecutive nucleotides defining a first binding domain of atleast 4 nucleotides;

[0053] (b) consecutive nucleotides defining a catalytic domain locatedcontiguous with the 3′ end of the first binding domain, and capable ofcleaving the PAI-1-encoding mRNA at a predetermined phosphodiester bond;and

[0054] (c) consecutive nucleotides defining a second binding domain ofat least 4 nucleotides located contiguous with the 3′ end of thecatalytic domain,

[0055] wherein the sequence of the nucleotides in each binding domain iscomplementary to a sequence of ribonucleotides in the PAI-1-encodingmRNA and wherein the catalytic nucleic acid hybridizes to andspecifically cleaves the PAI-1-encoding mRNA.

[0056] In different embodiments the catalytic deoxyribonucleic acidmolecules can cleave the PAI-1-encoding mRNA at the linkage between 5′-A(or G) and U(T)-3′ (or C) i.e. at purine:pyrimidine consensus sequences.In further embodiments the catalytic deoxyribonucleic acid moleculescleaves the PAI-1 mRNA at the linkage of any 5′-AU-3′; 5′-AC-3′;5′-GU-3′; or 5′-GC-3′ site within the mRNA.

[0057] To target the human PAI-1-encoding mRNA, catalytic nucleic acidscan be designed based on the consensus cleavage sites5′-purine:pyrimidine-3′ in the mRNA sequence (GenBank Accession#:M16006) (36). Those potential cleavage sites located on an open loop ofthe mRNA according to RNA folding software e.g. RNAdraw 2.1 areparticularly preferred as targets (22). The DNA based catalytic nucleicacids can utilize the structure where two sequence-specific arms areattached to a catalytic core (SEQ ID NO:1) based on the PAI-1-encodingmRNA sequence (corresponding DNA shown as SEQ ID NO:5). Further examplesof catalytic DNA structure are detailed in (23) and (24). Commerciallyavailable mouse brain polyA-RNA (Ambion) can serve as a template in thein vitro cleavage reaction to test the efficiency of the catalyticdeoxyribonucleic acids.

[0058] Catalytic nucleic acid molecules can cleave PAI-1-encoding mRNAat each and any of the consensus sequences therein. Since catalyticribo- and deoxyribo-nucleic acid consensus sequences are known, and thePAI-1-encoding mRNA sequence is known, one of ordinary skill couldreadily construct a catalytic ribo- or deoxyribo nucleic acid moleculedirected to any of the PAI-1-encoding mRNA consensus sequences based onthe instant specification. In preferred embodiments of this inventionthe catalytic deoxyribonucleic acids include the 10-23 structure.Cleavage of PAI-1 mRNA by DNAzyme may occur at 264 cleavage sites in thecoding region of human PAI-1-encoding mRNA, including 51 5′-AT-3′ (-AU-)sites, 76 -AC- sites, 53 -GT- (-GU-) sites and 84 -GC- sites. See FIGS.10 and 11 for AT and AC cleavage sites respectively, represented on theDNA sequence corresponding to the PAI-1-encoding mRNA.

[0059] Taking -AT- sites as a example, cleavage sites include nucleotide76, 82, 152, 159, 254, 277, 316, 328, 333, 340, 344, 355, 374, 391, 399,410, 415, 433, 449, 472, 543, 547, 550, 554, 583, 586, 644, 717, 745,748, 773, 794, 800, 811, 847, 853, 866, 901, 919, 939, 1027, 1036, 1120,1125, 1168, 1183, 1198, 1201, 1204, 1261, and 1273 of SEQ ID NO:5.

[0060] Examples of catalytic ribonucleic acids include hairpin andhammerhead ribozymes. In preferred embodiments of this invention, thecatalytic ribonucleic acid molecule is formed in a hammerhead (25) orhairpin motif (26,27,28), but may also be formed in the motif of ahepatitis delta virus (29), group I intron (35), RNaseP RNA (inassociation with an RNA guide sequence) (30,31) or Neurospora VS RNA(32,33,34). Hammerhead ribozymes can cleave any 5′-NUH-3′ triplets of amRNA, where U is conserved and N is any nucleotide and H can be C,U,A,but not G. For example, there are 151 sites which can be cleaved by ahammerhead ribozyme in human PAI-1-encoding mRNA coding region. See FIG.12.

[0061] Cleaving of PAI-1-encoding mRNA with catalytic nucleic acidsinterferes with one or more of the normal functions of PAI-1-encodingmRNA. The functions of mRNA to be interfered with include all vitalfunctions such as, for example, translocation of the RNA to the site ofprotein translation, translation of protein from the RNA, splicing ofthe RNA to yield one or more mRNA species, and catalytic activity whichmay be engaged in by the RNA.

[0062] In one embodiment the nucleotides of the first binding domaincomprise at least one deoxyribonucleotide. In another embodiment thenucleotides of the second binding domain comprise at least onedeoxyribonucleotide. In one embodiment the nucleotides of the firstbinding domain comprise at least one deoxyribonucleotide derivative. Inanother embodiment the nucleotides of the second binding domain compriseat least one deoxyribonucleotide derivative. In one embodiment thenucleotides of the first binding domain comprise at least oneribonucleotide. In another embodiment the nucleotides of the secondbinding domain comprise at least one ribonucleotide. In one embodimentthe nucleotides of the first binding domain comprise at least oneribonucleotide derivative. In another embodiment the nucleotides of thesecond binding domain comprise at least one ribonucleotide derivative.In one embodiment the nucleotides of the first binding domain compriseat least one modified base. In another embodiment the nucleotides of thesecond binding domain comprise at least one modified base.

[0063] The nucleotides may comprise other bases such as inosine,deoxyinosine, hypoxanthine may be used. In addition, isoteric purine2′deoxy-furanoside analogs, 2′-deoxynebularine or 2′deoxyxanthosine, orother purine or pyrimidine analogs may also be used. By carefullyselecting the bases and base analogs, one may fine tune thehybridization properties of the oligonucleotide. For example, inosinemay be used to reduce hybridization specificity, while diaminopurinesmay be used to increase hybridization specificity.

[0064] Adenine and guanine may be modified at positions N3, N7, N9, C2,C4, C5, C6, or C8 and still maintain their hydrogen bonding abilities.Cytosine, thymine and uracil may be modified at positions N1, C2, C4,C5, or C6 and still maintain their hydrogen bonding abilities. Some baseanalogs have different hydrogen bonding attributes than the naturallyoccurring bases. For example, 2-amino-2′-dA forms three (3), instead ofthe usual two (2), hydrogen bonds to thymine (T). Examples of baseanalogs that have been shown to increase duplex stability include, butare not limited to, 5-fluoro-2′-dU, 5-bromo-2′-dU, 5-methyl-2′-dC,5-propynyl-2′-dC, 5-propynyl-2′-dU, 2-amino-2′-dA, 7-deazaguanosine,7-deazadenosine, and N2-Imidazoylpropyl-2′-dG.

[0065] Nucleotide analogs may be created by modifying and/or replacing asugar moiety. The sugar moieties of the nucleotides may also be modifiedby the addition of one or more substituents. For example, one or more ofthe sugar moieties may contain one or more of the followingsubstituents: amino, alkylamino, araalkyl, heteroalkyl,heterocycloalkyl, aminoalkylamino, O, H, an alkyl, polyalkylamino,substituted silyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, S-alkyl,SOMe, SO₂Me, ONO₂, NH-alkyl, OCH₂CH═CH₂, OCH₂CCH, OCCHO, allyl, O-allyl,NO₂, N₃, and NH₂. For example, the 2′ position of the sugar may bemodified to contain one of the following groups: H, OH, OCN, O-alkyl, F,CN, CF₃, allyl, O-allyl, OCF₃, S-alkyl, SOMe, SO₂Me, ONO₂, NO₂, N₃, NH₂,NH-alkyl, or OCH═CH₂, OCCH, wherein the alkyl may be straight, branched,saturated, or unsaturated. In addition, the nucleotide may have one ormore of its sugars modified and/or replaced so as to be a ribose orhexose (i.e. glucose, galactose) or have one or more anomeric sugars.The nucleotide may also have one or more L-sugars.

[0066] Representative United States patents that teach the preparationof such modified bases/nucleosides/nucleotides include, but are notlimited to, U.S. Pat. Nos. 6,248,878, and 6,251,666 which are hereinincorporated by reference.

[0067] The sugar may be modified to contain one or more linkers forattachment to other chemicals such as fluorescent labels. In anembodiment, the sugar is linked to one or more aminoalkyloxy linkers. Inanother embodiment, the sugar contains one or more alkylamino linkers.Aminoalkyloxy and alkylamino linkers may be attached to biotin, cholicacid, fluorescein, or other chemical moieties through their amino group.

[0068] Nucleotide analogs or derivatives may have pendant groupsattached. Pendant groups serve a variety of purposes which include, butare not limited to, increasing cellular uptake of the molecule,enhancing degradation of the target nucleic acid, and increasinghybridization affinity. Pendant groups can be linked to the bindingdomains of the catalytic nucleic acid. Examples of pendant groupsinclude, but are not limited to: acridine derivatives (i.e.2-methoxy-6-chloro-9-aminoacridine); cross-linkers such as psoralenderivatives, azidophenacyl, proflavin, and azidoproflavin; artificialendonucleases; metal complexes such as EDTA-Fe(II),o-phenanthroline-Cu(I), and porphyrin-Fe(II); alkylating moieties;nucleases such as amino-1-hexanolstaphylococcal nuclease and alkalinephosphatase; terminal transferases; abzymes; cholesteryl moieties;lipophilic carriers; peptide conjugates; long chain alcohols; phosphateesters; amino; mercapto groups; radioactive markers; nonradioactivemarkers such as dyes; and polylysine or other polyamines. In oneexample, the nucleic acid comprises an oligonucleotide conjugated to acarbohydrate, sulfated carbohydrate, or gylcan. Conjugates may beregarded as a way as to introduce a specificity into otherwiseunspecific DNA binding molecules by covalently linking them to aselectively hybridizing oligonucleotide.

[0069] The binding domains of the catalytic nucleic acid may have one ormore of their sugars modified or replaced so as to be ribose, glucose,sucrose, or galactose, or any other sugar. Alternatively, they may haveone or more sugars substituted or modified in its 2′ position, i.e.2′allyl or 2′-O-allyl. An example of a 2′-O-allyl sugar is a2′-O-methylribonucleotide. Further, the nucleotides of the bindingdomain may have one or more of their sugars substituted or modified toform an α-anomeric sugar.

[0070] A catalytic nucleic acid binding domain may includenon-nucleotide substitution. The non-nucleotide substitution includeseither abasic nucleotide, polyether, polyamine, polyamide, peptide,carbohydrate, lipid or polyhydrocarbon compounds. The term “abasic” or“abasic nucleotide” as used herein encompasses sugar moieties lacking abase or having other chemical groups in place of base at the 1′position.

[0071] In one embodiment the nucleotides of the first binding domaincomprise at least one modified internucleoside bond. In anotherembodiment the nucleotides of the second binding domain comprise atleast one modified internucleoside bond. In a further embodiment themodified internucleoside bond is a phosphorothioate bond. The nucleicacid may comprise modified bonds. For example the bonds betweennucleotides of the catalytic nucleic acid may comprise phosphorothioatelinkages. The nucleic acid may comprise nucleotides having moiety may bemodified by replacing one or both of the two bridging oxygen atoms ofthe linkage with analogues such as —NH, —CH₂, or —S. Other oxygenanalogues known in the art may also be used. The phosphorothioate bondsmay be stereo regular or stereo random.

[0072] In one embodiment the PAI-1-encoding mRNA encodes human PAI-1. Ina further embodiment the human PAI-1-encoding mRNA has the sequence setforth in SEQ ID NO:5.

[0073] This invention provides a pharmaceutical composition comprisingthe instant catalytic nucleic acid and a pharmaceutically acceptablecarrier. The following pharmaceutically acceptable carriers are setforth, in relation to their most commonly associated delivery systems,by way of example, notwithstanding the fact that the instantpharmaceutical compositions are preferably delivered dermally.

[0074] Dermal delivery systems include, for example, aqueous andnonaqueous gels, creams, multiple emulsions, microemulsions, liposomes,ointments, aqueous and nonaqueous solutions, lotions, aerosols,hydrocarbon bases and powders, and can contain excipients such assolubilizers, permeation enhancers (e.g., fatty acids, fatty acidesters, fatty alcohols and amino acids), and hydrophilic polymers (e.g.,polycarbophil and polyvinylpyrolidone). In one embodiment, thepharmaceutically acceptable carrier is a liposome or a transdermalenhancer. Examples of liposomes which can be used in this inventioninclude the following: (1) CellFectin, 1:1.5 (M/M) liposome formulationof the cationic lipidN,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmity-spermine anddioleoyl phosphatidylethanolamine (DOPE)(GIBCO BRL); (2) Cytofectin GSV,2:1 (M/M) liposome formulation of a cationic lipid and DOPE (GlenResearch); (3) DOTAP(N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniumethyl sulfate)(Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposomeformulation of the polycationic lipid DOSPA and the neutral lipid DOPE(GIBCO BRL).

[0075] Transmucosal delivery systems include patches, tablets,suppositories, pessaries, gels and creams, and can contain excipientssuch as solubilizers and enhancers (e.g., propylene glycol, bile saltsand amino acids), and other vehicles (e.g., polyethylene glycol, fattyacid esters and derivatives, and hydrophilic polymers such ashydroxypropylmethylcellulose and hyaluronic acid).

[0076] Injectable drug delivery systems include solutions, suspensions,gels, microspheres and polymeric injectables, and can compriseexcipients such as solubility-altering agents (e.g., ethanol, propyleneglycol and sucrose) and polymers (e.g., polycaprylactones and PLGA's).Implantable systems include rods and discs, and can contain excipientssuch as PLGA and polycaprylactone.

[0077] Oral delivery systems include tablets and capsules. These cancontain excipients such as binders (e.g., hydroxypropylmethylcellulose,polyvinyl pyrilodone, other cellulosic materials and starch), diluents(e.g., lactose and other sugars, starch, dicalcium phosphate andcellulosic materials), disintegrating agents (e.g., starch polymers andcellulosic materials) and lubricating agents (e.g., stearates and talc).

[0078] This invention provides a method of specifically inhibiting theexpression of PAI-1 in a cell that would otherwise express PAI-1,comprising contacting the cell with the instant catalytic nucleic acidso as to specifically inhibit the expression of PAI-1 in the cell.

[0079] This invention provides a method of specifically inhibiting theexpression of PAI-1 in a subject's cells comprising administering to thesubject an amount of the instant catalytic nucleic acid effective tospecifically inhibit the expression of PAI-1 in the subject's cells.

[0080] This invention provides a method of specifically inhibiting theexpression of PAI-1 in a subject's cells comprising administering to thesubject an amount of the instant pharmaceutical composition effective tospecifically inhibit the expression of PAI-1 in the subject's cells.

[0081] This invention provides a method of treating a cardiovasculardisease in a subject involving apoptosis of a cardiomyocyte in thesubject which comprises administering to the subject an amount of theinstant pharmaceutical composition effective to inhibit apoptosis of thecardiomyocyte in the subject so as to thereby treat the cardiovasculardisease.

[0082] In one embodiment the cardiovascular disease is congestive heartfailure. In another embodiment the cardiovascular disease is myocardialinfarct. In another embodiment the cardiovascular disease is angina. Inanother embodiment the cardiovascular disease is myocardial ischemia. Inanother embodiment the cardiovascular disease is a cardiomyopathy.

[0083] This invention provides a method of treating a fibrotic diseasein a subject involving fibrogenesis which comprises administering to thesubject an amount of the instant pharmaceutical composition effective toinhibit fibrogenesis in the subject so as to thereby treat the fibroticdisease. In differing embodiments the fibrotic disease is a renaldisease, a hepatic disease, a disease of the lung, a disease of theskin, or a disease of the eye. In further embodiments the fibroticdisease of the skin is scleroderma or psorasis.

[0084] An “effective amount” is an amount at least sufficient to treat,reduce, reverse or otherwise inhibit the given disease state. In thecase of fibrotic diseases this means an amount sufficient to reduce,reverse or otherwise inhibit fibrogenesis. In the case of cardiovasculardiseases this means an amount sufficient to reduce, reverse or otherwiseinhibit cardiomyocyte apoptosis.

[0085] Determining the effective amount of the instant pharmaceuticalcompositions can be done based on animal data using routinecomputational methods. In one embodiment, the effective amount containsbetween about 10 ng and about 100 μg of the instant nucleic acidmolecules per kg body mass. In another embodiment, the effective amountcontains between about 100 ng and about 10 μg of the nucleic acidmolecules per kg body mass. In a further embodiment, the effectiveamount contains between about 1 μg and about 5 μg of the nucleic acidmolecules per kg body mass. In another embodiment the effective amountcontains between about 10 μg and about 100 μg of the nucleic acidmolecules per kg body mass. In a preferred embodiment the effectiveamount contains between about 100 μg and 500 μg of the nucleic acidmolecules per kg body mass. In another embodiment the effective amountcontains between about 500 μg and 1000 μg of the nucleic acid moleculesper kg body mass. In another embodiment the effective amount containsbetween about 1 mg and 2 mg of the nucleic acid molecules per kg bodymass.

[0086] The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes, receptortargeted molecules, oral, rectal, topical or other formulations, forassisting in uptake, distribution and/or absorption. RepresentativeUnited States patents that teach the preparation of such uptake,distribution and/or absorption assisting formulations include, but arenot limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

[0087] It is preferred to administer catalytic nucleic acids to mammalssuffering from a cardiovascular disease or a fibrotic disease, in eithernative form or suspended in a carrier medium in amounts and upontreatment schedules which are effective to therapeutically treat themammals to reduce the detrimental effects of cardiovascular disease orfibrotic disease. One or more different catalytic nucleic acids orantisense oligonucleotides or analogs thereof targeting differentsections of the nucleic acid sequence of PAI-1-encoding mRNA may beadministered together in a single dose or in different doses and atdifferent amounts and times depending upon the desired therapy. Thecatalytic nucleic acids can be administered to mammals in a mannercapable of getting the nucleic acids initially into the blood stream andsubsequently into cells, or alternatively in a manner so as to directlyintroduce the catalytic nucleic acids into the cells or groups of cells,for example cardiomyocytes, by such means by electroporation or bydirect injection into the heart or by catheter into renal tissue. It iswithin the scale of a person's skill in the art to determine optimumdosages and treatment schedules for such treatment regimens.

[0088] The effective amount of catalytic nucleic acid is administered toa human patient in need of inhibition of PAI-1 expression (or inhibitionof fibrogenesis) from 1-8 or more times daily or every other day. Dosageis dependent on severity and responsiveness of the effects of thecardiovascular or fibrotic disease to be treated, with a course oftreatment lasting from several days to months or until a cure iseffected or a reduction of the effects is achieved. The actual effectiveamount, or dosage, administered may take into account the size andweight of the patient, whether the nature of the treatment isprophylactic, therapeutic in nature, the age, weight, health and sex ofthe patient, the route of administration, and other factors.

[0089] Pharmaceutical compositions may contain suitable excipients andauxiliaries which facilitate processing of the catalytic nucleic acidsinto preparations which can be used pharmaceutically. Preferably, thepreparations, particularly those which can be administered orally andwhich can be used for the preferred type of administration, such astablets, dragees, and capsules, and preparations which can beadministered rectally, such as suppositories, as well as suitablesolutions for administration parenterally or orally, and compositionswhich can be administered bucally or sublingually, including inclusioncompounds, contain from about 0.1 to about 99 percent by weight ofactive ingredients, together with the excipient.

[0090] The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself well known in the art. Forexample, the pharmaceutical preparations may be made by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. The process to be used will depend ultimately onthe physical properties of the active ingredient used.

[0091] Suitable excipients are, in particular, fillers such as sugars,for example, lactose or sucrose, mannitol or sorbitol, cellulosepreparations and/or calcium phosphates, for example, tricalciumphosphate or calcium hydrogen phosphate as well as binders such asstarch, paste, using, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/orpolyvinyl pyrrolidone. If desired, disintegrating agents may be added,such as the above-mentioned starches as well as carboxymethyl-starch,cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a saltthereof, such as sodium alginate. Auxiliaries are flow-regulating agentsand lubricants, for example, such as silica, talc, stearic acid or saltsthereof, such as magnesium stearate or calcium stearate, and/orpolyethylene glycol. Dragee cores may be provided with suitable coatingswhich, if desired, may be resistant to gastric juices. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinylpyrrolidone, polyethylene, glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. In order to produce coatings resistant to gastricjuices, solutions of suitable cellulose preparations such asacetyl-cellulose phthalate or hydroxypropylmethycellulose phthalate, areused. Dyestuffs and pigments may be added to the tablets of drageecoatings, for example, for identification or in order to characterizedifferent combinations of active compound doses. Other pharmaceuticalpreparations which can be used orally include push-fit capsules made ofgelatin, as well as soft, sealed capsules made of gelatin and aplasticizer such as glycerol or sorbitol. The push-fit capsules cancontain the active compounds in the form of granules which may be mixedwith filters such as lactose, binders such as starches, and/orlubricants such as talc or magnesium stearate and optionally,stabilizers. In soft capsules, the active compounds are preferablydissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycols. In additions, stabilizers maybe added.

[0092] Possible pharmaceutical preparations which can be used rectallyinclude, for example, suppositories, which consist of a combination ofthe active compounds with a suppository base. Suitable suppository basesare, for example, natural or synthetic triglycerides, paraffinhydrocarbons, polyethylene glycols or higher alkanols. In addition, itis also possible to use gelatin rectal capsules which consist of acombination of the active compounds with a base. Possible base materialsinclude, for example liquid triglycerides, polyethylene glycols, orparaffin hydrocarbons.

[0093] Suitable formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble orwater-dispersible form. In addition, suspensions of the active compoundsas appropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, ethyloleate or triglycerides. Aqueous injection suspensions may containsubstances which increase the viscosity of the suspension including, forexample, sodium carboxymethyl cellulose, sorbitol, and/or dextran.Optionally, the suspension may also contain stabilizers.

[0094] Additionally, catalytic nucleic acids of the present inventionmay also be administered encapsulated in liposomes or immunoliposomes,which are pharmaceutical compositions wherein the active ingredient iscontained either dispersed or variously present in corpuscles consistingof aqueous concentric layers adherent to lipidic layers. Liposomes areespecially active in targeting the oligonucleotides to liver cells. Theactive ingredient, depending upon its solubility, may be present both inthe aqueous layer and in the lipidic layer, or in what is generallytermed a liposomic suspension. The hydrophobic layer, generally but notexclusively, comprises phospholipids such as lecithin and sphingomyelin,steroids such as cholesterol, more or less ionic surfactants such asdicetylphosphate, stearylamine, or phosphatidic acid, and/or othermaterials of a hydrophobic nature. The diameters of the liposomesgenerally range from about 15 nm to about 5 microns.

[0095] This invention also provides an oligonucleotide comprisingconsecutive nucleotides that hybridizes with a PAI-1-encoding mRNA underconditions of high stringency and is between 8 and 40 nucleotides inlength. In different embodiments the oligonucleotide is between 40 and80 nucleotides in length.

[0096] In a preferred embodiment the hybridization of antisenseoligonucleotides with PAI-1-encoding mRNA interferes with one or more ofthe normal functions of PAI-1-encoding mRNA. The functions of mRNA to beinterfered with include all vital functions such as, for example,translocation of the RNA to the site of protein translation, translationof protein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and catalytic activity which may be engaged in by the RNA. Forexample, a human PAI-1 antisense oligonucleotide specifically hybridizesunder given stringent conditions with targets on the human PAI-1 mRNAmolecule and in doing so inhibits the translation thereof into PAI-1protein.

[0097] The functions of mRNA to be interfered with include all vitalfunctions such as, for example, translocation of the RNA to the site ofprotein translation, translation of protein from the RNA, splicing ofthe RNA to yield one or more mRNA species, and catalytic activity whichmay be engaged in by the RNA. A PAI-1 antisense oligonucleotidespecifically hybridizes under given stringent conditions with targets onthe PAI-1-encoding mRNA molecule and in doing so inhibits thetranslation thereof into PAI-1.

[0098] In accordance with this invention, persons of ordinary skill inthe art will understand that messenger RNA includes not only theinformation to encode a protein using the three letter genetic code, butalso associated ribonucleotides which form a region known to suchpersons as the 5′-untranslated region, the 3′-untranslated region, the5′ cap region and intron/exon junction ribonucleotides. Thus, catalyticnucleic acids or antisense oligonucleotides may be formulated inaccordance with this invention which are targeted wholly or in part tothese associated ribonucleotides as well as to the informationalribonucleotides. For example, the antisense oligonucleotides maytherefore be specifically hybridizable with a transcription initiationsite region, a translation initiation codon region, a 5′ cap region, anintron/exon junction, coding sequences, a translation termination codonregion or sequences in the 5′- or 3′-untranslated region. Similarly, thecatalytic nucleic acids may specifically cleave a transcriptioninitiation site region, a translation initiation codon region, a 5′ capregion, an intron/exon junction, coding sequences, a translationtermination codon region or sequences in the 5′- or 3′-untranslatedregion. As is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule). A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterm “translation initiation codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine in eukaryotes. It is also known in the art that eukaryoticgenes may have two or more alternative translation initiation codons,any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the invention, “translationinitiation codon” refers to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding PAI-1, regardless of the sequence(s) of such codons. It is alsoknown in the art that a translation termination codon of a gene may haveone of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (thecorresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA,respectively). The term “translation initiation codon region” refers toa portion of such an mRNA or gene that encompasses from about 25 toabout 50 contiguous nucleotides in either direction (i.e., 5′ or 3′)from a translation initiation codon. This region is one preferred targetregion. Similarly, the term “translation termination codon region”refers to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation termination codon. This region is also onepreferred target region. The open reading frame or “coding region,”which is known in the art to refer to the region between the translationinitiation codon and the translation termination codon, is also a regionwhich may be targeted effectively. Other preferred target regionsinclude the 5′ untranslated region (5′UTR), known in the art to refer tothe portion of an mRNA in the 5′ direction from the translationinitiation codon, and thus including nucleotides between the 5′ cap siteand the translation initiation codon of an mRNA or correspondingnucleotides on the gene, and the 3′ untranslated region (3′UTR), knownin the art to refer to the portion of an mRNA in the 3′ direction fromthe translation termination codon, and thus including nucleotidesbetween the translation termination codon and 3′ end of an mRNA orcorresponding nucleotides on the gene. mRNA splice sites may also bepreferred target regions, and are particularly useful in situationswhere aberrant splicing is implicated in disease, or where anoverproduction of a particular mRNA splice product is implicated indisease. Aberrant fusion junctions due to rearrangements or deletionsmay also be preferred targets.

[0099] Once the target site or sites have been identified, antisenseoligonucleotides can be chosen which are sufficiently complementary tothe target, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired disruption of the function of themolecule. “Hybridization”, in the context of this invention, meanshydrogen bonding, also known as Watson-Crick base pairing, betweencomplementary bases, usually on opposite nucleic acid strands or tworegions of a nucleic acid strand. Guanine and cytosine are examples ofcomplementary bases which are known to form three hydrogen bonds betweenthem. Adenine and thymine are examples of complementary bases which formtwo hydrogen bonds between them. “Specifically hybridizable” and“complementary” are terms which are used to indicate a sufficient degreeof complementarity such that stable and specific binding occurs betweenthe DNA or RNA target and the antisense oligonucleotide. Similarly,catalytic nucleic acids are synthesized once cleavage target sites onthe PAI-1 mRNA molecule have been identified, e.g. any purine:pyrimidineconsensus sequences in the case of DNA enzymes. Catalytic nucleic acidsand antisense oligonucleotides targeted to disrupting polymorphisms andmutants of the PAI-1 maybe similarly made as described above based onthe polymorphic or mutant PAI-1 mRNA sequence.

[0100] Methods for selecting which particular antisense oligonucleotidessequences directed towards a particular protein-encoding mRNA are thatwill form the most stable DNA:RNA hybrids within the given target mRNAsequence are known in the art and are exemplified in U.S. Pat. No.6,183,966 which is herein incorporated by reference.

[0101] In one embodiment at least one internucleoside linkage within theinstant oligonucleotide comprises a phosphorothioate linkage. Antisenseoligonucleotide molecules synthesized with a phosphorothioate backbonehave proven particularly resistant to exonuclease damage compared tostandard deoxyribonucleic acids, and so they are used in preference. Aphosphorothioate antisense oligonucleotide for PAI-1 mRNA can besynthesized on an Applied Biosystems (Foster City, Calif.) model 380BDNA synthesizer by standard methods. For example, sulfurization can beperformed using tetraethylthiuram disulfide/acetonitrile. Followingcleavage from controlled pore glass support, oligodeoxynucleotides canbe base deblocked in ammonium hydroxide at 60° C. for 8 h and purifiedby reversed-phase HPLC [0.1M triethylammonium bicarbonate/acetonitrile;PRP-1 support]. Oligomers can be detritylated in 3% acetic acid andprecipitated with 2% lithiumperchlorate/acetone, dissolved in sterilewater and reprecipitated as the sodium salt from 1 M NaCl/ethanol.Concentrations of the full length species can be determined by UVspectroscopy. Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0102] Preferred modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3T-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included. Representative UnitedStates patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, each of which is herein incorporated by reference.

[0103] In one embodiment the nucleotides of the instant oligonucleoptidecomprise at least one deoxyribonucleotide. In another embodiment thenucleotides comprise at least one ribonucleotide. Suchdeoxyribonucleotides or ribonucleotides can be modified or derivatizedas described hereinabove.

[0104] In one embodiment the PAI-1-encoding mRNA encodes human PAI-1. Ina further embodiment the human PAI-1-encoding mRNA comprises consecutivenucleotides, the sequence of which is set forth in SEQ ID NO:5.

[0105] This invention further provides a method of treating a subjectwhich comprises administering to the subject an amount of the instantoligonucleotide effective to inhibit expression of a PAI-1 in thesubject so as to thereby treat the subject.

[0106] This invention further provides a method of treating acardiovascular disease in a subject involving apoptosis of acardiomyocyte in the subject which comprises administering to thesubject an amount of the instant oligonucleotide effective to inhibitapoptosis of the cardiomyocyte in the subject so as to thereby treat thecardiovascular disease.

[0107] This invention further provides a method of treating a fibroticdisease in a subject involving fibrogenesis in the subject whichcomprises administering to the subject an amount of the instantoligonucleotide effective to inhibit fibrogenesis in the subject so asto thereby treat the fibrotic disease.

[0108] Antisense oligonucleotides can be administered by intravenousinjection, intravenous drip, subcutaneous, intraperitoneal orintramuscular injection, orally or rectally. Human pharmacokinetics ofcertain antisense oligonucleotides have been studied. See (37)incorporated by reference in its entirety.

[0109] This invention provides all of the instant methods wherein thesubject is a mammal. In a preferred embodiment the mammal is a humanbeing.

[0110] In this invention, the various embodiments of subjects,pharmaceutically acceptable carriers, dosage, cell types, routes ofadministration and target nucleic acid sequences are envisioned for eachof the instant nucleic acid molecules, oligonucleotides, pharmaceuticalcompositions and methods.

[0111] This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

[0112] Experimental Results

[0113] Construction of DNA Enzymes Targeting Human and Rat PAI-1 mRNA.

[0114] We designed three DNA enzymes to specifically targetpyrimidine-purine junctions at or near the translational start site AUGof human and rat PAI-1 messenger RNA, a region that is conserved betweenspecies and has low relative free energy (17). As shown in FIG. 1a, thethree DNA enzymes, termed E1 (SEQ ID NO:2), E2 (SEQ ID NO:4), and E3(SEQ ID NO:3), contained identical 15-nucleotide catalytic domains (SEQID NO:1) which were flanked by two arms of eight nucleotides (E1, E2) ornine nucleotides (E3) with complementarity to human (E1, E3) or rat (E2)PAI-1 mRNA. To produce the control DNA enzyme E0 (SEQ ID NO:7), thenucleotide sequence in the two flanking arms of E2 was scrambled withoutaltering the catalytic domain (FIG. 1a). The 3′ terminus of eachmolecule was capped with an inverted 3′-3′-linked thymidine forresistance to 3′-to-5′ exonuclease digestion.

[0115] Specific Cleavage of Human PAI-1 mRNA by E1 and E3 DNA Enzymes.

[0116] As shown in FIG. 1b, the 21-base oligonucleotide S1 (SEQ IDNO:8), synthesized from human PAI-1 mRNA and labeled at the 5′ end with32P, was cleaved within 2 minutes when cultured with E1 at 10:1substrate:enzyme excess, with maximal cleavage occurring by 2 hours. The10-nucleotide cleavage product is consistent with the size of the32P-labeled fragment at the 5′ end. E1 also cleaved larger 32P-labeledfragments of human PAI-1 mRNA, prepared by in vitro transcription, in atime- and concentration-dependent manner, FIG. 1c. A 520 nucleotidetranscript was maximally cleaved by 2-4 hours to expected cleavageproducts of 320 and 200 nucleotides. A similar dose-dependency was seenwith E3, with maximal cleavage of human PAI-1 mRNA transcripts occurringat the highest concentration used, 20 μM. The sequence-specific natureof the DNA enzymatic cleavage is shown in FIG. 1d, where the control DNAenzyme E0, containing an identical catalytic domain to E1 and E3, butscrambled sequences in the flanking arms, caused no cleavage of humanPAI-1 mRNA transcripts. Pre-heating the transcript to 72° C. for 10minutes prior to incubation with E1, but not E0, further increasedcleavage.

[0117] Specific Cleavage of Rat PAI-1 mRNA by E2 DNA Enzyme.

[0118] Further evidence for the specificity of the DNA enzymaticreactions can be seen in FIG. 2. The DNA enzyme E2 cleaved the 23-baseoligonucleotide S2, synthesized from the sequence of rat PAI-1 mRNA, ina dose- and time-dependent manner, FIG. 2a. E2 also cleaved a rat PAI-1mRNA transcript in a dose-dependent manner by 2-4 hours to give the 156nucleotide cleavage product, FIG. 2b. In contrast, neither the scrambledcontrol DNA enzyme E0 nor E3 cleaved the rat PAI-1 mRNA transcript.Since the rat PAI-1 mRNA transcript (SEQ ID NO:10) differs by only onenucleotide from the human mRNA PAI-1 transcript (SEQ ID NO:9) which canbe cleaved by E3, FIG. 2c, these results demonstrate the exquisitetarget specificity of these DNA enzymes.

[0119] DNA Enzymes Inhibit Induction of Endogenous PAI-1 mRNA andProtein.

[0120] To determine the effect of DNA enzymes on endogenous PAI-1production, endothelial monolayers of human and rat origin were grown toconfluence and transfected with species-specific DNA enzymes orscrambled control. Transfected cells were then activated with TGF-betafor 8 hours to maximally induce expression of PAI-1. Densitometricanalysis of RT-PCR products following reverse transcription of cellularmRNA showed that E2 inhibited TGF-beta inducible steady-state mRNAlevels in cultured rat endothelium by 52%, FIG. 3a, relative to the E0scrambled DNA. Constitutive PAI-1 mRNA levels were not affected bytransfection with the DNA enzymes. FIG. 3b shows the effect ofendothelial cell transfection with E2 DNA enzyme on TGF-beta mediatedinduction of PAI-1 protein. Endothelial cells transfected with scrambledDNA enzyme demonstrated approximately 50% increase in cytoplasmic PAI-1protein as detected by Western blot. In contrast, this effect was almostcompletely is abrogated by transfection with the PAI-1 DNA enzyme E2.

[0121] Resistance of DNA Enzymes to Serum-Dependent Degradation.

[0122] Since DNA enzymes with a thymidine in the correct orientation atthe 3′ end are significantly degraded by factors in serum (1-5%concentration) within 24 hours (14), we investigated the protectiveeffect of an inverted thymidine at the 3′ end on serum-dependentnucleolytic degradation of PAI-1 DNA enzymes. The DNA enzyme E1 waslabeled with 32P and incubated for 3-24 hours with human umbilical veinendothelial cell (HUVEC) monolayers cultured in medium containingphysiologic (2%) or supraphysiologic (20%) serum concentrations As shownin FIG. 3c, whereas the DNA enzyme was almost completely degraded within6 hours of cell culture in medium containing 20% serum, it remainedintact during the entire 24 hours of cell culture with medium containing2% serum. As shown in FIG. 3d, HUVEC transfected with E1 or E3 DNAenzymes and cultured for 12 hours in 2% serum demonstrated 20% and 28%reduction in TGF-beta dependent PAI-1 activity compared withtransfection with the scrambled DNA E0 (both p<0.01). Together, theseresults indicate that an inverted thymidine at the 3′ end can protectthe PAI-1 DNA enzyme against nucleolytic degradation at physiologicserum concentrations likely to be encountered in vivo, consequentlyenabling inhibition of cellular PAI-1 activity.

[0123] Intracardiac Injection of E2 Reduces Macrophage PAI-1 Expression.

[0124] We investigated whether intracardiac injection of E2 at the timeof myocardial infarction could inhibit PAI-1 expression in the ratheart. At two weeks after ligation of the left anterior descending (LAD)artery and induction of myocardial infarction, immunohistochemicalstudies showed that PAI-1 expression occurred predominantly inCD68-positive rat macrophages within the infarct zone and at theperi-infarct region. While a few vascular endothelial cells stainedpositively, PAI-1 expression was not detected within myocytes orinterstitial cells distally to the infarct. We conclude that theprincipal source of PAI-1 protein in the infarcted rat heart is due toproduction by infiltrating macrophages and phagocytes. At two weeksafter injection with E2, PAI-1 expression in infarcted rat hearts wasdramatically altered, with 71% reduction in PAI-1 expression bymacrophages at the peri-infarct region (p<0.01), FIG. 4a. Inhibition ofPAI-1 expression at this site was highly specific since PAI-1 expressionwithin macrophages in the center of the infarct zone remained unaffectedafter E2 injection, and injection with the scrambled DNA enzyme E0 didnot reduce peri-infarct PAI-1 expression.

[0125] Intracardiac Injection of E2 Results in Peri-InfarctNeovascularization.

[0126] By two weeks, in parallel with reduction in PAI-1 expression,hearts from rats injected with E2 demonstrated significantly increasednumbers of large-lumen capillaries (>6 nuclei) at the peri-infarctregion, FIG. 4b. While injection of E2 alone caused a significantincrease in large-lumen capillaries at the peri-infarct region comparedwith injection of the E0 scrambled DNA enzyme, even more striking wasthe additional 62% increase in the number of large-lumen capillarieswhen E2 was co-administered together with intravenously delivered humanadult bone marrow-derived CD34+CD117^(bright) angioblasts. While many ofthe newly-formed vessels were of rat origin (angiogenesis), asignificant number were of human origin (vasculogenesis), as defined bya human anti-CD31 mAb. In the presence of E2 the number ofunincorporated, discrete, interstitial human angioblasts in the infarctzone and peri-infarct region was reduced by 5.2-fold compared with E0control enzyme, FIG. 4c. Conversely, the number of large-lumencapillaries (>6 nuclei) at the peri-infarct region was increased by14-fold following E2 injection as compared with E0. We conclude that byinhibiting PAI-1 expression at the peri-infarct region E2 enables humanangioblasts to more effectively migrate through cardiac tissue, coalesceand participate in new vessel formation.

[0127] E2 Protects Cardiomyocytes at the Peri-Infarct Region AgainstApoptosis.

[0128] We have previously shown that neovascularization at theperi-infarct region, induced by bone marrow-derived endothelialprogenitors, can protect adjacent viable cardiomyocytes againstapoptosis. Since intracardiac injection of E2 alone inducedneovascularization at this site in infarcted rat hearts, we examined theeffect of E2 injection on cardiomyocyte apoptosis. Injection with E2 atthe time of LAD ligation resulted in 70% reduction in apoptosis ofcardiomyocytes at the peri-infarct region (p<0.01), FIG. 5a. This wasnot observed with injection of the scrambled control DNA enzyme E0. Thelevel in protection against cardiomyocyte apoptosis was similar to thelevel observed with intravenous infusion of human angioblasts.

[0129] Renal Fibrosis.

[0130] The renin-angiotensin system (RAS) in progressive renal diseasehas been extensively investigated, indicating multiple actions beyondhemodynamic and salt/water homeostasis. The RAS is now recognized to belinked to induction of plasminogen activator inhibitor-1 (PAI-1) likelyvia both the type 1 (AT1) and type 4 (AT4) receptors, thus, promotingboth thrombosis and fibrosis (39). Plasminogen activator inhibitor type1 is thus a suitable target in renal fibrogenesis. The progression ofrenal lesions to fibrosis involves several mechanisms, among which theinhibition of extracellular matrix (ECM) degradation appears to play animportant role. Two interrelated proteolytic systems are involved inmatrix degradation: the plasminogen activation system and the matrixmetalloproteinase system. PAI-1 as the main inhibitor of plasminogenactivation, regulates fibrinolysis and the plasmin-mediated matrixmetalloproteinase activation. PAI-1 is also a component of the ECM,where it binds to vitronectin. PAI-1 is not expressed in the normalhuman kidney but is strongly induced in various forms of kidneydiseases, leading to renal fibrosis and terminal renal failure.Thrombin, angiotensin II, and transforming growth factor-beta are potentin vitro and in vivo agonists in increasing PAI-1 synthesis. Severalexperimental and clinical studies support a role for PAI-1 in the renalfibrogenic process occurring in chronic glomerulonephritis, diabeticnephropathy, focal segmental glomerulosclerosis, and other fibroticrenal diseases (40).

[0131] End-stage renal disease (ESRD) comprises an enormous publichealth burden, with an incidence and prevalence that are increasingly onthe rise. This escalating prevalence suggests that newer therapeuticinterventions and strategies are needed to complement currenttherapeutic approaches. Although much evidence demonstrates conclusivelythat angiotensin II mediates progressive renal disease, recent evidencealso implicates aldosterone as an important pathogenetic factor inprogressive renal disease. Recently, several lines of experimentalevidence demonstrate that selective blockade of aldosterone, independentof renin-angiotensin blockade, reduces proteinuria and nephrosclerosisin the spontaneously hypertensive stroke-prone rat (SHRSP) model andreduces proteinuria and glomerulosclerosis in the subtotallynephrectomized rat model (ie, remnant kidney). Whereas pharmacologicblockade with angiotensin II receptor blockers andangiotensin-converting enzyme (ACE) inhibitors reduces proteinuria andnephrosclerosis/glomerulosclerosis, selective reinfusion of aldosteronerestores these abnormalities despite continued renin-angiotensinblockade. Aldosterone may promote fibrosis by several mechanisms,including plasminogen activator inhibitor-1 (PAI-1) expression andconsequent alterations of vascular ribrinolysis, by stimulation oftransforming growth factor-betal (TGF-betal), and by stimulation ofreactive oxygen species (ROS) (41). Therefore it is expected that PAI-1is a suitable target for therapeutic intervention into renal fibrosis.

[0132] Progressive renal disease is characterized by the induction ofplasminogen activator inhibitor-1 (PAI-1), suggesting that impairedactivity of the renal plasmin cascade may play a role in renal fibrosis.Studies with PAI-1 knock-out mice showing resistance to fibrotic effectsof ureteric obstruction establish an important fibrogenic role for PAI-1in the renal fibrogenic response. The results demonstrate that oneimportant fibrosis-promoting function of PAI-1 is its role in therecruitment of fibrosis-inducing cells, including myofibroblasts andmacrophages (42).

[0133] Liver Fibrosis.

[0134] During chronic liver injury, transforming growth factor beta(TGF-beta) plays a prominent role in stimulating liver fibrogenesis bymyofibroblast-like cells derived from hepatic stellate cells (HSCs). Inacute liver injury, HSC-derived TGF-beta increased plasminogen activatorinhibitor type 1 (PAI-1) and alpha2(I) procollagen (COL1A2) transcripts.Smad 2 in HSCs during liver injury and primary cultured HSCs wereactivated by an autocrine mechanism, because high levels of Smad 2phosphorylation and induction of PAI-1 transcript by TGF-beta wereobserved in HSCs (43).

[0135] Increased PAI-1 together with uPA, uPAR and tPA are associatedwith overall inhibition of matrix degradation in cirrhotic liver.Hepatic stellate cells are an important source of PAI-1 during liverfibrosis. Increased expression of plasminogen activator and plasminogenactivator inhibitor during liver fibrogenesis of rats: role of stellatecells (44). Therefore it is expected that PAI-1 is a suitable target fortherapeutic intervention into hepatic fibrosis.

[0136] Lung Fibrosis:

[0137] PAI-1 levels in bronchoalveolar lavage (BAL) supernatant fluidsare significantly higher in idiopathic pulmonary fibrosis (IPF) patientsthan in normal subjects. Increased procoagulant and antifibrinolyticactivities in the lungs with idiopathic pulmonary fibrosis. Therefore itis expected that PAI-1 is a suitable target for therapeutic interventioninto fibrosis of the lung.

[0138] In addition, cataracts of the eye may be treated by reversing thesymptomatic fibrosis that occurs, thought to be due to ischemia, by themechanism described hereinabove.

[0139] Materials and Methods

[0140] DNA enzymes and RNA substrates: DNA enzymes with 3′-3′ invertedthymidine were synthesized by Integrated DNA technologies (Coralville,Iowa) and purified by RNase-free IE-HPLC or RP-HPLC. The short RNAsubstrates corresponding to target DNA enzyme sequences were chemicallysynthesized followed by RNAse-free PAGE purification and also made by invitro transcription from a DNA template.

[0141] In vitro transcription: Human PAI-1 cDNA was amplified by RT-PCRfrom total RNA of cultured HUVEC using the following primer pair:5′CCAAGAGCGCTGTCAAGAAGAC3′ (forward primer; SEQ ID NO:11) and5′TCACCGTCTGCTTTGGAGACCT3′ (reverse primer; SEQ ID NO:12) (position25-10600, J. Biol. Chem, 1988, 263(19): 9143). Length of PCR product is1598 bases. PAI-1 cDNA was cloned into pGEM-T vector (Promega) to obtainplasmid construct pGEM-hPAI. cDNA sequence was verified using anautomatic sequencing machine. A 32P-labeled-nucleotide human PAI-1 RNAtranscript was prepared by in vitro transcription (SP6 polymerase,Promega). 20 ml transcription reaction consisted of 4 μl 5× buffer, 2 μlDTT, 1 μl RNasin inhibitor, 4 μl NTP mixture (1 μl A,G,C, and 1 ul H₂O),100 μM UTP, 2 μl template (0.3 μg/μl), a-32P-UTP (10 μci/μl) and 1 μlSP6 polymerase (20 u/μl). Reaction time was 1 hour at 32° C.Unincorporated label and short nucleotides (<350base) were seperatedfrom radiolabeled species by centrigugation on Chromaspin-200 columns(Clontech, Palo Alto, Calif.).

[0142] Cleavage reactions: synthetic RNA substrate was end-labeled with32P using T4 polynucleotide kinase. Cleavage reaction system included 60mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 150 mM NaCl, 0.5 μM 32p-labled RNAoligo., 0.05-5 μM DNA enzyme. For cleavage of in vitro transcripts,reaction system contained 1% of PAI-1 transcripts, 25 mM Tris-HCl (pH7.5), 5 mM MgCl₂, 100 mM NaCl and 0.2-20 μM DNA enzyme. Reactions wereallowed to proceed at 37° C. and were “quenched ” by transfer ofaliquots to tubes containing 90% formamide, 20 mM EDTA and loading dye.Samples were separated by electrophoresis on TBE-urea denaturingpolyacrylamide gels (5% gel for in vitro transcripts and 15% forsynthetic RNA) and detected by autoradiography at −80° C.

[0143] DNA enzyme stability in serum: DAN enzymes were radiolabled usingT4 polynucleotide kinase. Labeling reaction (20 μl of volume) consistsof 1 μl 20 μM DNA enzyme, 2 μl 10× kinase buffer, 1 μl T4 PNK (10 u/μl),10 μl H₂O, and 6 μl 32P-ATP (3000 uCi/mmol, 10 uCi/μl). Reaction time is30 minutes at 37° C. HUVEC were cultured in media (Clonetic) containing2% or 20% FCS, and radiolabeled oligomers were added at a finalconcentration of 100 nM. Aliquots of the mixture were removed atdifferent time points of 0, 3, 6, 12, 24 hours, quenched withphenol/chloroform and frozen until use. At the end of each experiment,all samples were phenol-extracted and analyzed by 15% denaturingpolyacrylamide gels and visualized by autoradiography.

[0144] Culture conditions and DNA enzyme transfection: Primary HUVECendothelial cells were obtained from Clonetic (USA) and grown in mediumcontaining 2%FCS, 100 ug/ml streptomycin and 100 IU/ml penicillin at 37°C. in a humidified atmosphere of 5% CO2. HUVEC were used in experimentsbetween passage 6 and 8. For transfection of DNA enzyme, rat endothelialcells (EC) were harvested and seeded into each well of 6-well plates(˜1×10⁵ cells/well). Subconfluent (70-80%) EC were washed twice with 1ml HEPES buffer (pH 7.4) and transfected using 0.5 ml of serum-freemedium containing 1 μM test molecule (E2 or E0) and 20 μg/ml cationiclipids (DOTAP). 3 hours after incubation, 0.5 ml of 2% serum medium wereadded to each well; 3 hours later, TGFb was added to half the wells at afinal concentration of 1.8 ng/ml. Transfected cells continued to beincubated for 8 hrs and were lysed to isolate RNA (for RT-PCR) andProtein (for Western blot) using Trizol reagent (Life Technologies).

[0145] RT-PCR: Total RNA from each well of 6-well plates were isolatedusing 1 ml Trizol reagent and dissolved in 20 μl Depc-treated H₂O. 2 μlsamples were used to perform reverse transcription in 20 μl reactionsystem, and 4 μl products were then used as templates to amplify PAI-1or human GAPDH in 50 μl PCR system (5 μl 10× buffer, 1 μl dNTP, 0.25 μlTaq polymerase, 1 μl forward and reverse primer, 38 μl H₂O, 2 μl 32pdCTP(3000 ci/mmol, 10 uci/ul). The reaction conditions were 95° C.-2 min(predenatured), 95° C.-0.5 min, 58° C.-1 min, 72° C.-2 min, 35 cycles,72° C.-10 min. Human GAPDH was used as internal control (forward Primer5′TGAAGGTCGGAGTCAACGGATTTG3′; SEQ ID NO:13, reverse primer5′CATGTGGGCCATGAGGTCCACCAC3′; SEQ ID NO:14), its PCR products being 452base.

[0146] Western blot: Control and DNA enzyme-treated cells wereharvested, washed with PBS and then the cell pellets were resuspended inlysis buffer and processed. Equal amounts of protein (15 μg/lane) wereseparated by SDS gel electrophoresis with 10% polyacrylamine separatinggel using minigels (Bio-Rad). After electrophoresis, proteins weretransferred to nitrocellulose membrane (Protran, Schleicler & Schuell)by electrotransfer and blocked for at least one hour at room temperaturewith 5% (W/V) BSA in TBS-T buffer (0.1M Tris-base (pH 7.5), 0.15M Nacland 0.1% Tween-20) Following this step, membranes were immunoblottedwith goat IgG polyclonal anti-PAI antibodies (Santa Cruz biotech) andthen visualised by the ECL-system (Amershan) using horseradishperoxidase conjugated anti-goat IgG (Sigma).

[0147] Measurement of plasmin activity (PAI-1 functional assay):Conditioned medium and cell lysates were collected at various timesafter interventions. The cells were washed twice with PBS and lysed in400 ml of 0.5% Triton X-100 for assaying plasmin. Conditioned media andTriton cell lysates were incubated with plasmin substrateVal-Leu-Lys-pNA (S2251, Kabivitru, Stockholm, Sweden) (finalconcentration, 0.35 mM), in a 96-well microtiter plate (final volume,200 μl) for 60 min at room temperature. Plasmin activity was measured byreading the absorbance at 410 nm and was calculated against a plasmin(Sigma) standard regression line.

[0148] Animals, surgical procedures, injection of human cells, andquantitation of cellular migration into tissues: Rowett (rnu/rnu)athymic nude rats (Harlan Sprague Dawley, Indianapolis, Ind.) were usedin studies approved by the “Columbia University Institute for AnimalCare and Use Committee”. After anesthesia, a left thoracotomy wasperformed, the pericardium was opened, and the left anterior descending(LAD) coronary artery was ligated. At the time of surgery one group ofrats received three intracardiac injections of E2 DNA enzyme, anotherreceived three intracardiac injections of E0 scrambled control, and athird group received three intracardiac injections of saline. Forstudies on neovascularization, 2.0×10⁶ human cells obtained from asingle donor after G-CSF mobilization were reconstituted with 2.0×10⁵immunopurified CD34+^(CD117bright) cells, and injected into the rat tailvein 48 hours after LAD ligation. Each group consisted of 6-10 rats.

[0149] Quantitation of capillary density: In order to quantitatecapillary density and species origin of the capillaries, additionalsections were stained freshly with mAbs directed against factor VIII,rat or human CD31 (all Serotec, UK), and rat or human MHC class I(Accurate Chemicals, CT). Staining was performed by immunoperoxidasetechnique using an Avidin/Biotin Blocking Kit, a rat-absorbedbiotinylated anti-mouse IgG, and a peroxidase-conjugate (all VectorLaboratories Burlingame, Calif.). Capillary density was determined fromsections labeled with anti-Factor VIII mAb at 2 weeks post infarctionand compared to the capillary density of the unimpaired myocardium.Values are expressed as factor VIII positive cells per HPF (600×).

[0150] Measurement of myocyte apoptosis by DNA end-labeling of paraffintissue sections: For in situ detection of apoptosis at the single celllevel we used the TUNEL method of DNA end-labeling mediated bydexynucleotidyl transferase (TdT) (Boehringer Mannheim, Mannheim,Germany). Rat myocardial tissue sections were obtained from LAD-ligatedrats at two weeks after injection of either saline or CD34+ human cells,and from healthy rats as negative controls. Briefly, tissues weredeparaffinized with xylene and rehydrated with graded dilutions ofethanol and two washes in phosphate-buffered saline (PBS). The tissuesections were then digested with Proteinase K (10 μg/ml in Tris/HCL) for30 minutes at 37° C. The slides were then washed 3 times in PBS andincubated with 50 μl of the TUNEL reaction mixture (TdT andfluorescein-labeled dUTP) and incubated in a humid atmosphere for 60minutes at 37° C. For negative controls TdT was eliminated from thereaction mixture. Following 3 washes in PBS, the sections were thenincubated for 30 minutes with an antibody specific forfluorescein-conjugated alkaline phosphatase (AP) (Boehringer Mannheim,Mannheim, Germany). The TUNEL stain was visualized with a substratesystem in which nuclei with DNA fragmentation stained blue, (BCIP/NBTsubstrate system, DAKO, Carpinteria, Calif.). The reaction wasterminated following three minutes of exposure with PBS. To determinethe proportion of blue-staining apoptotic nuclei within myocytes, tissuewas counterstained with a monoclonal antibody specific for desmin.Endogenous peroxidase was blocked by using a 3% hydrogen perioxidasesolution in PBS for 15 minutes, followed by washing with 20% goat serumsolution. An anti-desmin antibody (Sigma, Saint Louis, Mo.) wasincubated overnight (1:100) at 40° C. Following 3 washes sections werethen treated with an anti-rabbit IgG, followed by a biotin conjugatedsecondary antibody for 30 minutes (Sigma, Saint Louis, Mo.). Anavidin-biotin complex (Vector Laboratories, Burlingame, Calif.) was thenadded for an additional 30 minutes and the myocytes were visualizedbrown following 5 minutes exposure in DAB solution mixture (Sigma, SaintLouis, Mo.). Tissue sections were examined microscopically at 40×magnification and at least 100 cells were counted in a minimum of 8high-power fields. The percentage of apoptotic myocytes was determinedby means of an apoptotic index; the apoptotic index was calculated bydividing the number of positive staining myocyte nuclei by the totalnumber of myocyte nuclei and multiplying by 100. Stained cells at theedges of the tissue were not counted. An apoptotic index of 1 or lesswas considered to indicate the absence of apoptosis.

[0151] Analyses of myocardial function: Echocardiographic studies wereperformed using a high frequency liner array transducer (SONOS 5500,Hewlett Packard, Andover, Mass.). 2D images were obtained atmid-papillary and apical levels. End-diastolic (EDV) and end-systolic(ESV) left ventricular volumes were obtained by bi-plane area-lengthmethod, and % left ventricular ejection fraction was calculated as[(EDV-ESV)/EDV]×100. Cardiac output (CO) was measured using anultrasonic flowprobe (Transonic Systems Inc., Ithaca, N.Y.) and cardiacindex was calculated as CO per weight. All echocardiographic studieswere performed by a blinded investigator (ST).

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[0197]

1 17 1 15 DNA ARTIFICIAL SEQUENCE DNAzyme Catalytic Domain 1 ggctagctacaacga 15 2 31 DNA ARTIFICIAL SEQUENCE DNAzyme for human PlasminogenActivator Inhibitor-1 2 catctgcagg ctagctacaa cgacctgaag t 31 3 33 DNAARTIFICIAL SEQUENCE DNAzyme for human Plasminogen Activator Inhibitor-13 ctggagacag gctagctaca acgactgcat cct 33 4 31 DNA ARTIFICIAL SEQUENCEDNAzyme for Plasminogen Activator Inhibitor 4 gctgaagagg ctagctacaacgaatctgca t 31 5 2876 DNA Homo Sapiens 5 gaattcctgc agctcagcagccgccgccag agcaggacga accgccaatc gcaaggcacc 60 tctgagaact tcaggatgcagatgtctcca gccctcacct gcctagtcct gggcctggcc 120 cttgtctttg gtgaagggtctgctgtgcac catcccccat cctacgtggc ccacctggcc 180 tcagacttcg gggtgagggtgtttcagcag gtggcgcagg cctccaagga ccgcaacgtg 240 gttttctcac cctatggggtggcctcggtg ttggccatgc tccagctgac aacaggagga 300 gaaacccagc agcagattcaagcagctatg ggattcaaga ttgatgacaa gggcatggcc 360 cccgccctcc ggcatctgtacaaggagctc atggggccat ggaacaagga tgagatcagc 420 accacagacg cgatcttcgtccagcgggat ctgaagctgg tccagggctt catgccccac 480 ttcttcaggc tgttccggagcacggtcaag caagtggact tttcagaggt ggagagagcc 540 agattcatca tcaatgactgggtgaagaca cacacaaaag gtatgatcag caacttgctt 600 gggaaaggag ccgtggaccagctgacacgg ctggtgctgg tgaatgccct ctacttcaac 660 ggccagtgga agactcccttccccgactcc agcacccacc gccgcctctt ccacaaatca 720 gacggcagca ctgtctctgtgcccatgatg gctcagacca acaagttcaa ctatactgag 780 ttcaccacgc ccgatggccattactacgac atcctggaac tgccctacca cggggacacc 840 ctcagcatgt tcattgctgccccttatgaa aaagaggtgc ctctctctgc cctcaccaac 900 attctgagtg cccagctcatcagccactgg aaaggcaaca tgaccaggct gccccgcctc 960 ctggttctgc ccaagttctccctggagact gaagtcgacc tcaggaagcc cctagagaac 1020 ctgggaatga ccgacatgttcagacagttt caggctgact tcacgagtct ttcagaccaa 1080 gagcctctcc acgtcgcgcaggcgctgcag aaagtgaaga tcgaggtgaa cgagagtggc 1140 acggtggcct cctcatccacagctgtcata gtctcagccc gcatggcccc cgaggagatc 1200 atcatggaca gacccttcctctttgtggtc cggcacaacc ccacaggaac agtccttttc 1260 atgggccaag tgatggaaccctgaccctgg ggaaagacgc cttcatctgg gacaaaactg 1320 gagatgcatc gggaaagaagaaactccgaa gaaaagaatt ttagtgttaa tgactctttc 1380 tgaaggaaga gaagacatttgccttttgtt aaaagatggt aaaccagatc tgtctccaag 1440 accttggcct ctccttggaggacctttagg tcaaactccc tagtctccac ctgagaccct 1500 gggagagaag tttgaagcacaactccctta aggtctccaa accagacggt gacgcctgcg 1560 ggaccatctg gggcacctgcttccacccgt ctctctgccc actcgggtct gcagacctgg 1620 ttcccactga ggccctttgcaggatggaac tacggggctt acaggagctt ttgtgtgcct 1680 ggtagaaact atttctgttccagtcacatt gccatcactc ttgtactgcc tgccaccgcg 1740 gaggaggctg gtgacaggccaaaggccagt ggaagaaaca ccctttcatc tcagagtcca 1800 ctgtggcact ggccacccctccccagtaca ggggtgctgc aggtggcaga gtgaatgtcc 1860 cccatcatgt ggcccaactctcctggcctg gccatctccc tccccagaaa cagtgtgcat 1920 gggttatttt ggagtgtaggtgacttgttt actcattgaa gcagatttct gcttcctttt 1980 atttttatag gaatagaggaagaaatgtca gatgcgtgcc cagctcttca ccccccaatc 2040 tcttggtggg gaggggtgtacctaaatatt tatcatatcc ttgcccttga gtgcttgtta 2100 gagagaaaga gaactactaaggaaaataat attatttaaa ctcgctccta gtgtttcttt 2160 gtggtctgtg tcaccgtatctcaggaagtc cagccacttg actggcacac acccctccgg 2220 acatccagcg tgacggagcccacactgcca ccttgtggcc gcctgagacc ctcgcgcccc 2280 ccgcgccccc cgcgcccctctttttcccct tgatggaaat tgaccataca atttcatcct 2340 ccttcagggg atcaaaaggacggagtgggg ggacagagac tcagatgagg acagagtggt 2400 ttccaatgtg ttcaatagatttaggagcag aaatgcaagg ggctgcatga cctaccagga 2460 cagaactttc cccaattacagggtgactca cagccgcatt ggtgactcac ttcaatgtgt 2520 catttccggc tgctgtgtgtgagcagtgga cacgtgaggg gggggtgggt gagagagaca 2580 ggcagctcgg attcaactaccttagataat atttctgaaa acctaccagc cagagggtag 2640 ggcacaaaga tggatgtaatgcactttggg aggccaaggc gggaggattg cttgagccca 2700 ggagttcaag accagcctgggcaacatacc aagacccccg tctctttaaa aatatatata 2760 ttttaaatat acttaaatatatatttctaa tatctttaaa tatatatata tattttaaag 2820 accaatttat gggagaattgcacacagatg tgaaatgaat gtaatctaat agaagc 2876 6 402 PRT HOMO SAPIENS 6Met Gln Met Ser Pro Ala Leu Thr Cys Leu Val Leu Gly Leu Ala Leu 1 5 1015 Val Phe Gly Glu Gly Ser Ala Val His His Pro Pro Ser Tyr Val Ala 20 2530 His Leu Ala Ser Asp Phe Gly Val Arg Val Phe Gln Gln Val Ala Gln 35 4045 Ala Ser Lys Asp Arg Asn Val Val Phe Ser Pro Tyr Gly Val Ala Ser 50 5560 Val Leu Ala Met Leu Gln Leu Thr Thr Gly Gly Glu Thr Gln Gln Gln 65 7075 80 Ile Gln Ala Ala Met Gly Phe Lys Ile Asp Asp Lys Gly Met Ala Pro 8590 95 Ala Leu Arg His Leu Tyr Lys Glu Leu Met Gly Pro Trp Asn Lys Asp100 105 110 Glu Ile Ser Thr Thr Asp Ala Ile Phe Val Gln Arg Asp Leu LysLeu 115 120 125 Val Gln Gly Phe Met Pro His Phe Phe Arg Leu Phe Arg SerThr Val 130 135 140 Lys Gln Val Asp Phe Ser Glu Val Glu Arg Ala Arg PheIle Ile Asn 145 150 155 160 Asp Trp Val Lys Thr His Thr Lys Gly Met IleSer Asn Leu Leu Gly 165 170 175 Lys Gly Ala Val Asp Gln Leu Thr Arg LeuVal Leu Val Asn Ala Leu 180 185 190 Tyr Phe Asn Gly Gln Trp Lys Thr ProPhe Pro Asp Ser Ser Thr His 195 200 205 Arg Arg Leu Phe His Lys Ser AspGly Ser Thr Val Ser Val Pro Met 210 215 220 Met Ala Gln Thr Asn Lys PheAsn Tyr Thr Glu Phe Thr Thr Pro Asp 225 230 235 240 Gly His Tyr Tyr AspIle Leu Glu Leu Pro Tyr His Gly Asp Thr Leu 245 250 255 Ser Met Phe IleAla Ala Pro Tyr Glu Lys Glu Val Pro Leu Ser Ala 260 265 270 Leu Thr AsnIle Leu Ser Ala Gln Leu Ile Ser His Trp Lys Gly Asn 275 280 285 Met ThrArg Leu Pro Arg Leu Leu Val Leu Pro Lys Phe Ser Leu Glu 290 295 300 ThrGlu Val Asp Leu Arg Lys Pro Leu Glu Asn Leu Gly Met Thr Asp 305 310 315320 Met Phe Arg Gln Phe Gln Ala Asp Phe Thr Ser Leu Ser Asp Gln Glu 325330 335 Pro Leu His Val Ala Gln Ala Leu Gln Lys Val Lys Ile Glu Val Asn340 345 350 Glu Ser Gly Thr Val Ala Ser Ser Ser Thr Ala Val Ile Val SerAla 355 360 365 Arg Met Ala Pro Glu Glu Ile Ile Met Asp Arg Pro Phe LeuPhe Val 370 375 380 Val Arg His Asn Pro Thr Gly Thr Val Leu Phe Met GlyGln Val Met 385 390 395 400 Glu Pro 7 30 DNA ARTIFICIAL SEQUENCESCRAMBLED CONTROL SEQUENCE 7 acactggagg ctagcacaac gatgacgagt 30 8 21RNA Homo Sapiens 8 aacuucagga ugcagauguc u 21 9 21 RNA Homo Sapiens 9aggaugcaga ugucuccagc c 21 10 24 RNA Rattus Sp. 10 gaugcagaug ucuucagcccucau 24 11 22 DNA ARTIFICIAL SEQUENCE PRIMER derived from human source11 ccaagagcgc tgtcaagaag ac 22 12 22 DNA ARTIFICIAL SEQUENCE PRIMERderived from human source 12 tcaccgtctg ctttggagac ct 22 13 24 DNAARTIFICIAL SEQUENCE PRIMER derived from human source 13 tgaaggtcggagtcaacgga tttg 24 14 24 DNA ARTIFICIAL SEQUENCE PRIMER derived fromhuman source 14 catgtgggcc atgaggtcca ccac 24 15 3053 DNA RattusNorvegicus 15 cccccgagag ctttgtgaag gaggaacgct gcacacccgc ctcccgcagcacacagccaa 60 ccacagctga gcgacacgca acaagagcca atcacaaggc acttccgaaagctccaggat 120 gcagatgtct tcagccctca cttgcctcac cctgggcctg gttctggtctttgggaaagg 180 gttcgcttca ccccttccag agtcccatac agcccagcag gccaccaacttcggagtaaa 240 agtgtttcag catgtggtcc aggcctccaa agaccgaaat gtggtcttctctccctacgg 300 cgtgtcctcg gtgctggcta tgctgcagct gaccacagca gggaaaacccggcagcagat 360 ccaagatgct atgggattca atatcagtga gaggggcaca gctcctgccctccgaaagct 420 ctccaaggag ctcatggggt catggaacaa gaatgagatc agtactgcggacgccatctt 480 tgtccagcgg gacctagagc tggtccaggg cttcatgccc cacttcttcaagctcttccg 540 gaccacggtg aagcaggtgg acttctcaga ggtggaaaga gccagattcatcatcaacga 600 ctgggtggag aggcacacca aaggtatgat cagtgactta ctggccaagggggctgtaaa 660 tgagctgaca cgcctggtgc tggtgaacgc cctctatttc aacggccaatggaagacccc 720 cttcttagag gccagcaccc accagcgcct gttccacaag tctgatggtagcaccatctc 780 cgtgcccatg atggctcaga acaacaagtt caactacact gagttcaccactccggatgg 840 gcacgagtac gacatcctgg aactgcccta ccacggcgaa accctcagcatgttcattgc 900 agcacccttt gaaaaagatg tgcccctctc cgccatcacc aacattttggacgctgagct 960 catcagacaa tggaagagca acatgaccag gctgccccgc ctcctcatcctgcctaagtt 1020 ctctctggag actgaagtgg acctcagagg gcccctggag aagctgggcatgactgacat 1080 cttcagctca acccaggccg acttcacaag tctttccgac caagagcagctctctgtagc 1140 acaagcacta caaaaggtca agatcgaggt gaacgagagc ggcacagtggcgtcttcctc 1200 cacagccatt ctagtctcag cccgcatggc ccccacggag atggttttagaccgatcctt 1260 tctctttgtg gttcggcaca atccaacaga gacaatcctc ttcatgggccagctgatgga 1320 gccttgagag tgggatgaga agcctttcct ttgggacaaa actggacgtgttataagcag 1380 agactctgaa gaaaagaatt gttttaagga ctctttgggg agaaagagaaggcctttctt 1440 tcttaccccg gcactggtaa atctttccaa ccagcctccc agacctcagactctcgaaga 1500 ggaaagagtc taactccctc actagggacc tatcttacta aggtctcatccaaccataga 1560 actcacagaa tctggatctg cccagcattc agcctttgga cccagttcccaccaaggccc 1620 cagcagggcc aacccactac gccttcactc agcaaagtct tttgtgttccagtcacactc 1680 tgggtacctc ttgtatcgtc ctccattgct atgaaggatg acccaggccaaaggaagaag 1740 cactgtccta tctcaaggtc cactgtggaa atgaacacct tgcccatccccaaggggcag 1800 cagatagaca gatcgaatga tcgcccgata tcaagccttc tcccagctcccgtcctgccc 1860 tcccttccct gacagccgcc ttgtgttatt tcagagtgta gatgacttgtttacagcttt 1920 tttcgaccca caaacttttc tcattttgaa agcgtgaaag aaaggtcagatgtgcacgtg 1980 ccttgctctt tatcctgggt ctccctgtga ggggagaggg gtcctggggagattccaggg 2040 gtgtgattga atatttatct tgtttatctt atacgtttgt tggggagaagaagcactatt 2100 aaggagaaag ccttttattt aaaccatggc atatggtgtc ccatttggggtctgtatccc 2160 tgtatgtcag ggaggcatca ctccacaaac ccgcccctcg ggtggcccggcgtcggggct 2220 cacactgccg cctagtggca gccgaacacg cccttgcccc atccctcccccgcatcctcc 2280 cccgtggctc ttttccttag ggatcttgcc aaggtgatgc ttggcagcccacggtaaagg 2340 aagggggaaa aagattaggt gggagagaga gagagagaga gagagagagagagagagaga 2400 gagagagaga gagagagaga gagagagaga aagagagaga gatgtttgagagagggcaaa 2460 gtggtttcaa atttttccaa tacattcaga agccgagtgg gaaagggggctgtgtgacct 2520 aacaggacag aactttctcc aattactggg tgactcagct gcactggtgactcacttcaa 2580 tgtgtcattt ccggctgctg taagtgagca gtggacacgt gggggggggggggtgaggat 2640 gaaagaaaca gccagctcct ggtcaaccac cttagttaga taatcttttttgaaagcttc 2700 ctagctgggg gtatgatcag aaaaccaatt tactgaaaaa ctgcacaggaaggtaacgtg 2760 aatctaattt catagcgggc cgctctgcat ccgttacatc tccactggaaaaaaataatc 2820 attttctttt tgtgtgtgtg tgtgtgtttt agcttttctc cctctccctctttctctctc 2880 atttcattat gcactggata accatacacc gtgtaccaca ggggcccaaatgtggggtca 2940 catggtcttg aattttgtgg ggtacatatg cctttgtttg tttgttttcacttttgatat 3000 ataaacaggt aaatgtgttt ttaaaaaata ataaaaatag agaatatgcagac 3053 16 402 PRT Rattus Norvegicus 16 Met Gln Met Ser Ser Ala Leu ThrCys Leu Thr Leu Gly Leu Val Leu 1 5 10 15 Val Phe Gly Lys Gly Phe AlaSer Pro Leu Pro Glu Ser His Thr Ala 20 25 30 Gln Gln Ala Thr Asn Phe GlyVal Lys Val Phe Gln His Val Val Gln 35 40 45 Ala Ser Lys Asp Arg Asn ValVal Phe Ser Pro Tyr Gly Val Ser Ser 50 55 60 Val Leu Ala Met Leu Gln LeuThr Thr Ala Gly Lys Thr Arg Gln Gln 65 70 75 80 Ile Gln Asp Ala Met GlyPhe Asn Ile Ser Glu Arg Gly Thr Ala Pro 85 90 95 Ala Leu Arg Lys Leu SerLys Glu Leu Met Gly Ser Trp Asn Lys Asn 100 105 110 Glu Ile Ser Thr AlaAsp Ala Ile Phe Val Gln Arg Asp Leu Glu Leu 115 120 125 Val Gln Gly PheMet Pro His Phe Phe Lys Leu Phe Arg Thr Thr Val 130 135 140 Lys Gln ValAsp Phe Ser Glu Val Glu Arg Ala Arg Phe Ile Ile Asn 145 150 155 160 AspTrp Val Glu Arg His Thr Lys Gly Met Ile Ser Asp Leu Leu Ala 165 170 175Lys Gly Ala Val Asn Glu Leu Thr Arg Leu Val Leu Val Asn Ala Leu 180 185190 Tyr Phe Asn Gly Gln Trp Lys Thr Pro Phe Leu Glu Ala Ser Thr His 195200 205 Gln Arg Leu Phe His Lys Ser Asp Gly Ser Thr Ile Ser Val Pro Met210 215 220 Met Ala Gln Asn Asn Lys Phe Asn Tyr Thr Glu Phe Thr Thr ProAsp 225 230 235 240 Gly His Glu Tyr Asp Ile Leu Glu Leu Pro Tyr His GlyGlu Thr Leu 245 250 255 Ser Met Phe Ile Ala Ala Pro Phe Glu Lys Asp ValPro Leu Ser Ala 260 265 270 Ile Thr Asn Ile Leu Asp Ala Glu Leu Ile ArgGln Trp Lys Ser Asn 275 280 285 Met Thr Arg Leu Pro Arg Leu Leu Ile LeuPro Lys Phe Ser Leu Glu 290 295 300 Thr Glu Val Asp Leu Arg Gly Pro LeuGlu Lys Leu Gly Met Thr Asp 305 310 315 320 Ile Phe Ser Ser Thr Gln AlaAsp Phe Thr Ser Leu Ser Asp Gln Glu 325 330 335 Gln Leu Ser Val Ala GlnAla Leu Gln Lys Val Lys Ile Glu Val Asn 340 345 350 Glu Ser Gly Thr ValAla Ser Ser Ser Thr Ala Ile Leu Val Ser Ala 355 360 365 Arg Met Ala ProThr Glu Met Val Leu Asp Arg Ser Phe Leu Phe Val 370 375 380 Val Arg HisAsn Pro Thr Glu Thr Ile Leu Phe Met Gly Gln Leu Met 385 390 395 400 GluPro 17 1209 DNA HOMO SAPIENS 17 atgcagatgt ctccagccct cacctgcctagtcctgggcc tggcccttgt ctttggtgaa 60 gggtctgctg tgcaccatcc cccatcctacgtggcccacc tggcctcaga cttcggggtg 120 agggtgtttc agcaggtggc gcaggcctccaaggaccgca acgtggtttt ctcaccctat 180 ggggtggcct cggtgttggc catgctccagctgacaacag gaggagaaac ccagcagcag 240 attcaagcag ctatgggatt caagattgatgacaagggca tggcccccgc cctccggcat 300 ctgtacaagg agctcatggg gccatggaacaaggatgaga tcagcaccac agacgcgatc 360 ttcgtccagc gggatctgaa gctggtccagggcttcatgc cccacttctt caggctgttc 420 cggagcacgg tcaagcaagt ggacttttcagaggtggaga gagccagatt catcatcaat 480 gactgggtga agacacacac aaaaggtatgatcagcaact tgcttgggaa aggagccgtg 540 gaccagctga cacggctggt gctggtgaatgccctctact tcaacggcca gtggaagact 600 cccttccccg actccagcac ccaccgccgcctcttccaca aatcagacgg cagcactgtc 660 tctgtgccca tgatggctca gaccaacaagttcaactata ctgagttcac cacgcccgat 720 ggccattact acgacatcct ggaactgccctaccacgggg acaccctcag catgttcatt 780 gctgcccctt atgaaaaaga ggtgcctctctctgccctca ccaacattct gagtgcccag 840 ctcatcagcc actggaaagg caacatgaccaggctgcccc gcctcctggt tctgcccaag 900 ttctccctgg agactgaagt cgacctcaggaagcccctag agaacctggg aatgaccgac 960 atgttcagac agtttcaggc tgacttcacgagtctttcag accaagagcc tctccacgtc 1020 gcgcaggcgc tgcagaaagt gaagatcgaggtgaacgaga gtggcacggt ggcctcctca 1080 tccacagctg tcatagtctc agcccgcatggcccccgagg agatcatcat ggacagaccc 1140 ttcctctttg tggtccggca caaccccacaggaacagtcc ttttcatggg ccaagtgatg 1200 gaaccctga 1209

What is claimed is:
 1. A catalytic nucleic acid that specificallycleaves an mRNA encoding a Plasminogen Activator Inhibitor-1 (PAI-1)comprising, in 5′ to 3′ order: (a) consecutive nucleotides defining afirst binding domain of at least 4 nucleotides; (b) consecutivenucleotides defining a catalytic domain located contiguous with the 3′end of the first binding domain, and capable of cleaving thePAI-1-encoding mRNA at a predetermined phosphodiester bond; and (c)consecutive nucleotides defining a second binding domain of at least 4nucleotides located contiguous with the 3′ end of the catalytic domain,wherein the sequence of the nucleotides in each binding domain iscomplementary to a sequence of ribonucleotides in the PAI-1-encodingmRNA and wherein the catalytic nucleic acid hybridizes to andspecifically cleaves the PAI-1-encoding mRNA.
 2. The catalytic nucleicacid of claim 1, wherein the nucleotides of the first binding domaincomprise at least one deoxyribonucleotide.
 3. The catalytic nucleic acidof claim 1, wherein the nucleotides of the second binding domaincomprise at least one deoxyribonucleotide.
 4. The catalytic nucleic acidof claim 1, wherein the nucleotides of the first binding domain compriseat least one deoxyribonucleotide derivative.
 5. The catalytic nucleicacid of claim 1, wherein the nucleotides of the second binding domaincomprise at least one deoxyribonucleotide derivative.
 6. The catalyticnucleic acid of claim 1, wherein the nucleotides of the first bindingdomain comprise at least one ribonucleotide.
 7. The catalytic nucleicacid of claim 1, wherein the nucleotides of the second binding domaincomprise at least one ribonucleotide.
 8. The catalytic nucleic acid ofclaim 1, wherein the nucleotides of the first binding domain comprise atleast one ribonucleotide derivative.
 9. The catalytic nucleic acid ofclaim 1, wherein the nucleotides of the second binding domain compriseat least one ribonucleotide derivative.
 10. The catalytic nucleic acidof claim 1, wherein the nucleotides of the first binding domain compriseat least one modified base.
 11. The catalytic nucleic acid of claim 1,wherein the nucleotides of the second binding domain comprise at leastone modified base.
 12. The catalytic nucleic acid of claim 1, whereinthe nucleotides of the first binding domain comprise at least onemodified internucleoside bond.
 13. The catalytic nucleic acid of claim1, wherein the nucleotides of the second binding domain comprise atleast one modified internucleoside bond.
 14. The catalytic nucleic acidof claim 12 or 13, wherein the modified internucleoside bond is aphosphorothioate bond.
 15. The catalytic nucleic acid of claim 1,wherein the PAI-1-encoding mRNA encodes human PAI-1.
 16. The catalyticnucleic acid of claim 15, wherein the human PAI-1-encoding mRNA has thesequence set forth in SEQ ID NO:5.
 17. A pharmaceutical compositioncomprising the catalytic nucleic acid of claim 1 and a pharmaceuticallyacceptable carrier.
 18. A method of specifically inhibiting theexpression of PAI-1 in a cell that would otherwise express PAI-1,comprising contacting the cell with the catalytic nucleic acid of claim1 so as to specifically inhibit the expression of PAI-1 in the cell. 19.A method of specifically inhibiting the expression of PAI-1 in asubject's cells comprising administering to the subject an amount of thecatalytic nucleic acid of claim 1 effective to specifically inhibit theexpression of PAI-1 in the subject's cells.
 20. A method of specificallyinhibiting the expression of PAI-1 in a subject's cells comprisingadministering to the subject an amount of the pharmaceutical compositionof claim 17 effective to specifically inhibit the expression of PAI-1 inthe subject's cells.
 21. A method of treating a cardiovascular diseasein a subject involving apoptosis of a cardiomyocyte in the subject whichcomprises administering to the subject an amount of the pharmaceuticalcomposition of claim 17 effective to inhibit apoptosis of thecardiomyocyte in the subject so as to thereby treat the cardiovasculardisease.
 22. A method of treating a fibrotic disease in a subjectinvolving fibrogenesis which comprises administering to the subject anamount of the pharmaceutical composition of claim 17 effective toinhibit fibrogenesis in the subject so as to thereby treat the fibroticdisease.
 23. The method of claim 22, wherein the fibrotic disease is arenal disease.
 24. The method of claim 22, wherein the fibrotic diseaseis a hepatic disease.
 25. The method of claim 22, wherein the fibroticdisease is a disease of the lung.
 26. The method of claim 22, whereinthe fibrotic disease is a disease of the skin.
 27. The method of claim22, wherein the fibrotic disease is a disease of the eye.
 28. Anoligonucleotide comprising consecutive nucleotides that hybridizes witha PAI-1-encoding mRNA under conditions of high stringency and is between8 and 40 nucleotides in length.
 29. The oligonucleotide of claim 28,wherein at least one internucleoside linkage within the oligonucleotidecomprises a phosphorothioate linkage.
 30. The oligonucleotide of claim28, wherein the nucleotides comprise at least one deoxyribonucleotide.31. The oligonucleotide of claim 28, wherein the nucleotides comprise atleast one ribonucleotide.
 32. The oligonucleotide of claim 28, whereinthe PAI-1-encoding mRNA encodes human PAI-1.
 33. The oligonucleotide ofclaim 32, wherein the human PAI-1-encoding mRNA comprises consecutivenucleotides, the sequence of which is set forth in SEQ ID NO:5.
 34. Amethod of treating a subject which comprises administering to thesubject an amount of the oligonucleotide of claim 28 effective toinhibit expression of a PAI-1 in the subject so as to thereby treat thesubject.
 35. A method of treating a cardiovascular disease in a subjectinvolving apoptosis of a cardiomyocyte in the subject which comprisesadministering to the subject an amount of the oligonucleotide of claim28 effective to inhibit apoptosis of the cardiomyocyte in the subject soas to thereby treat the cardiovascular disease.
 36. A method of treatinga fibrotic disease in a subject involving fibrogenesis in the subjectwhich comprises administering to the subject an amount of theoligonucleotide of claim 28 effective to inhibit fibrogenesis in thesubject so as to thereby treat the fibrotic disease.
 37. The method ofany of claim 19, 20, 21, 22, 34, 35, or 36, wherein the subject is amammal.
 38. The method of claim 37, wherein the mammal is a human being.